Evidence for Alpha7 Nicotinic Receptor Activation During the Cough Suppressing Effects Induced by Nicotine and Identification of ATA-101 as a Potential Novel Therapy for the Treatment of Chronic Cough.

Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α 4 ß 2)-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α 7-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α 7) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α 7 nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α 7 nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α 7 nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α7 nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α7 nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.

Pharmacological characterization of GSK573719 (umeclidinium): a novel, long-acting, inhaled antagonist of the muscarinic cholinergic receptors for treatment of pulmonary diseases.

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 µg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 µg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.

In vitro and in vivo pharmacological profile of PL-3994, a novel cyclic peptide (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)) natriuretic peptide receptor-A agonist that is resistant to neutral endopeptidase and acts as a bronchodilator.

The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (K(i)s of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC(50)s of 2, 3 and 14 nm). PL-3994 has a K(i) of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 µm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC(50)s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm-100 µm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1-1000 µg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.

The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo.

Lipoxins (LXs) and aspirin-triggered LX (ATL) are trihydroxytetraene-containing eicosanoids generated from arachidonic acid that are distinct in structure, formation, and function from the many other proinflammatory lipid-derived mediators. These endogenous eicosanoids have now emerged as founding members of the first class of lipid/chemical mediators involved in the resolution of the inflammatory response. Lipoxin A(4) (LXA(4)), ATL, and their metabolic stable analogs elicit cellular responses and regulate leukocyte trafficking in vivo by activating the specific receptor, ALX. ALX was the first receptor cloned and identified as a G protein-coupled receptor (GPCR) for lipoxygenase-derived eicosanoids with demonstrated cell type-specific signaling pathways. ALX at the level of DNA has sequence homology to the N-formylpeptide receptor and as an orphan GPCR was initially referred to as the N-formylpeptide receptor-like 1. Although LXA(4) is the endogenous potent ligand for ALX activation, a number of peptides can also activate this receptor to stimulate calcium mobilization and chemotaxis in vitro. In contrast with LXA(4), the counterparts of many of these peptides in vivo remain to be established. The purpose of this review is to highlight the molecular characterization of the ALX receptor and provide an overview of the ALX-LXA(4) axis responsible for anti-inflammatory and proresolving signals in vivo. The information in this review provides further support for the initial nomenclature proposition for this GPCR as ALX.

A novel role for tachykinin neurokinin-3 receptors in regulation of human bronchial Ganglia neurons.

The neuropeptide tachykinins and their receptors have been implicated in the pathogenesis of lung disease, although the role of the tachykinin neurokinin-3 receptor has not been elucidated. Using confocal microscopy, we identified tachykinin neurokinin-3 receptors on human bronchial parasympathetic ganglion neurons. Electrophysiologic recordings demonstrated that activation of sensory nerve fibers, either by antidromic stimulation or capsaicin, depolarized these neurons. This response was mimicked by exogenously applied tachykinin neurokinin-3 receptor-selective agonist, senktide analogue, but not significantly by tachykinin neurokinin-1 or neurokinin-2 receptor-selective agonists. Responses to endogenous tachykinins or exogenous selective tachykinin neurokinin-3 receptor activation with senktide analogue were inhibited by the selective tachykinin neurokinin-3 receptor antagonists, SB 223412 or SB 235375. We provide the first evidence that tachykinin neurokinin-3 receptors regulate human bronchial parasympathetic ganglion neurotransmission by activation of a peripheral reflex. This pathway may play a significant role in controlling bronchomotor tone and air flow to the lung.

International Union of Pharmacology XLIV. Nomenclature for the oxoeicosanoid receptor.

Oxoeicosanoids are a family of biologically active arachidonic acid derivatives that have been intimately linked with cellular migration. These metabolites are not only potent chemotaxins but also elicit oxygen radical production as well as induce secretory events in different cells. The most potent native ligand reported is 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), and the cell membrane receptor activated has now been cloned. This receptor is distinct from those receptors activated by either the prostaglandins or the leukotrienes. The purpose of this review is to briefly summarize the molecular evidence and highlight the significance of this receptor. In addition, an official nomenclature for this oxoeicosanoid receptor is proposed.

International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors.

The leukotrienes and lipoxins are biologically active metabolites derived from arachidonic acid. Their diverse and potent actions are associated with specific receptors. Recent molecular techniques have established the nucleotide and amino acid sequences and confirmed the evidence that suggested the existence of different G-protein-coupled receptors for these lipid mediators. The nomenclature for these receptors has now been established for the leukotrienes. BLT receptors are activated by leukotriene B(4) and related hydroxyacids and this class of receptors can be subdivided into BLT(1) and BLT(2). The cysteinyl-leukotrienes (LT) activate another group called CysLT receptors, which are referred to as CysLT(1) and CysLT(2). A provisional nomenclature for the lipoxin receptor has also been proposed. LXA(4) and LXB(4) activate the ALX receptor and LXB(4) may also activate another putative receptor. However this latter receptor has not been cloned. The aim of this review is to provide the molecular evidence as well as the properties and significance of the leukotriene and lipoxin receptors, which has lead to the present nomenclature.

A potent and selective nonpeptide antagonist of CXCR2 inhibits acute and chronic models of arthritis in the rabbit.

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.

Nonpeptide tachykinin receptor antagonists. III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyper-reactivity in guinea pigs.

In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited (125)I-[MePhe(7)]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K(i) = 2.2 nM and antagonized competitively NKB-induced Ca(2+) mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K(b) = 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA(2) = 8.1) and guinea pig ileal circular smooth muscles (pA(2) = 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 209 nM), and was without effect, at 1 microM, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED(50) of 0.56 mg/kg). Intraperitoneal SB 235375 (10-30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.